This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

Microarray and real-time RT-PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC-STAMP. Specific siRNA and antibodies were used to inhibit OC-STAMP RNA and protein, respectively, and TRAP+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/− RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL-induced differentiation was studied. OC-STAMP was very strongly up-regulated during osteoclast differentiation. Northern blots and sequence analysis revealed 2 transcripts of 2 kb and 3.7 kb differing only in 3'UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multi-pass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy-terminal 193 amino acids, however, are significantly similar to the DC-STAMP family consensus sequence. DC-STAMP is a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC-STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of tartrate-resistant acid phosphatase (TRAP) +, multinucleated cells in differentiating osteoclast cultures, with many TRAP + mononuclear cells present. Conversely, overexpression of OC-STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC-STAMP is a previously unknown, RANKL-induced, multi-pass transmembrane protein that promotes the formation of multinucleated osteoclasts.

The toothless (tl) mutation in the rat is a naturally occurring, autosomal recessive mutation resulting in a profound deficiency of bone-resorbing osteoclasts and peritoneal macrophages. The failure to resorb bone produces severe, unrelenting osteopetrosis, with a highly sclerotic skeleton, lack of marrow spaces, failure of tooth eruption, and other pathologies. Injections of CSF-1 improve some, but not all, of these. In this report we have used polymorphism mapping, sequencing, and expression studies to identify the genetic lesion in the tl rat. We found a 10-base insertion near the beginning of the open reading of the Csf1 gene that yields a truncated, nonfunctional protein and an early stop codon, thus rendering the tl rat CSF-1 null . All mutants were homozygous for the mutation and all carriers were heterozygous. No CSF-1 transcripts were identified in rat mRNA that would avoid the mutation via alternative splicing. The biology and actions of CSF-1 have been elucidated by many studies that use another naturally occurring mutation, the op mouse, in which a single base insertion also disrupts the reading frame. The op mouse has milder osteoclastopenia and osteopetrosis than the tl rat and recovers spontaneously over the first few months of life. Thus, the tl rat provides a second model in which the functions of CSF-1 can be studied. Understanding the similarities and differences in the phenotypes of these two models will be important to advancing our knowledge of the many actions of CSF-1.O steoclasts, multinucleated cells that resorb bone, differentiate via fusion of mononuclear precursors of the monocyte͞macrophage lineage, in large part under the local control of factors secreted by bone-forming osteoblasts (1). Insights into factors that regulate the formation and activation of osteoclasts have come from naturally occurring mutations and genetic manipulations that cause osteopetrosis, a condition in which defective bone resorption leads to a sclerotic skeleton (2). One such factor is the cytokine CSF-1 (M-CSF), originally identified as a monocyte-macrophage colony-stimulating factor (3). Identification of a frameshift mutation in the Csf1 gene of the osteopetrosis (op) mouse was a major step in understanding osteoclast ontogeny (4). (The op mutation in the mouse, which affects an osteoblast signal, is not to be confused with the op mutation in the rat, a severe, naturally occurring osteopetrotic mutation that affects osteoclast function.) Since then, many genes have been identified that do double-duty in the immune and skeletal systems. Tumor necrosis factor superfamily member 11 [TNFSF11, also called TRANCE (5), RANKL (6), ODF (7), and OPGL (8)], its receptor (RANK), and the receptorassociated intracellular signal initiator (TRAF6) are essential for lymph node organogenesis, maintaining antigen presenting dendritic cells, and formation of osteoclasts (refs. 9-12; reviewed in ref. 13). The transcription factor PU.1 functions in osteoclast differentiation and activation and in myeloid cells and B lymphocyt...

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-B ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1 tl /csf1 tl ) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.The catabolic removal of bone during skeletal formation and remodeling requires the specialized activity of multinucleated osteoclasts. Osteoclasts differentiate by fusion of hematopoietic mononuclear precursors in response to systemic and local signals, in particular colony-stimulating factor-1 (CSF-1, 2 or M-CSF) and the tumor necrosis factor family member receptor activator of NF-B ligand (RANKL, or TRANCE, TNFSF11) (1). Excessive osteoclast activity systemically leads to osteopenias such as osteoporosis, whereas local hyperactivity can lead to osteolysis as seen in tumor metastases to bone or in prosthesis loosening. Hypoactivity of osteoclasts can lead to sclerosing bone disorders, for example in genetic conditions such as osteopetrosis.Osteoclast differentiation is a complex process that requires the coordinated action of many gene products, including not only extrinsic factors such as CSF-1 and RANKL, which are supplied by osteoblasts locally in bone tissue, but also intrinsic factors required for osteoclast function. Mononuclear precursors must migrate to sites where resorption is needed, fuse to form multinucleated pre-osteoclasts, and attach firmly to bone. They develop highly specialized cellular structures, including an actin ring that forms a tight seal with the bone surface, and a highly convoluted plasma membrane domain called the ruffled border, which is the site of extremely a...

Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colonystimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor B).

No abstract

No abstract

We report the skeletal manifestations of restoring RANKL (TNFSF11/TRANCE; see foot note on nomenclature) expression in null mice using a lymphocyte-specific promoter. RANKL was discovered independently by immunologists and bone researchers by virtue of its essential roles in lymph node organogenesis, normal cellular immunity, and osteoclastogenesis. "Rescue" of RANKL knockout mice by a T- and B-cell expressed transgene reversed many immunological manifestations of the knockout, while it had highly selective effects on the skeletal pathology. RANKL-null mice exhibit severe osteopetrosis, no tooth eruption, markedly reduced skeletal growth, and growth plate chondrodystrophy. The transgene induced tartrate-resistant acid phosphatase (TRAP) positive cells in long bones as early as 3 days postpartum, restored marrow spaces in long bones, produced lamellar bone in the diaphyses, and restored osteoclasts at many endosteal sites, but not in periosteum nor the jaws. It did not improve the chondrodystrophy, chondroosseous junction defects, or tooth eruption. The ends of limb and axial skeletal elements remained highly sclerotic while diaphyses became osteopenic, and growth retardation persisted. Together, these results demonstrate the importance of local delivery of RANKL for many skeletal processes.